1. Field of Invention
This invention relates to microfluidic devices. In particular, the invention relates to microfluidic devices and their uses and methods for assaying cells.
2. Description of Related Art
Single cells represent a fundamental biological unit. However, the vast majority of biological knowledge has emerged as a consequence of studying cell populations and not individual cells. Inevitably, there are fundamental and applied questions, such as those relating to transcriptional control of stem cell differentiation, intrinsic noise in gene expression, and the origins of disease, that may only be addressed at the single cell level. For example, single cell analysis allows for the direct measurement of gene expression kinetics, or for the unambiguous identification of co-regulated genes, even in the presence of de-synchronization and heterogeneity that could obscure population-averaged measurements. Similarly, single cell methods are vital in stem cell research and cancer biology, where isolated populations of primary cells are heterogeneous due to limitations in purification protocols, and it is often a minority cell population that is the most relevant. High-throughput single cell measurement technologies are therefore of interest and have broad applications in clinical and research settings.
Existing methods for measuring transcript levels in single cells include RT-qPCR (1), single molecule counting using digital PCR (2) or hybridization probes (3, 4), and next generation sequencing (5). Of these, single cell RT-qPCR provides combined advantages of sensitivity, specificity, and dynamic range, but is limited by low throughput, high reagent cost, and difficulties in accurately measuring low abundance transcripts (6).
Microfluidic devices employing active valving to position and isolate cells have allowed for the isolation and genome amplification of individual microbial cells (32). Unfortunately, such devices do not allow for high throughput analysis due to the manual effort involved in operating the valving mechanisms. Moreover, the device does not allow isolated cells to be washed from the supernatant prior to treatment or analysis. This in turn allows for contamination events, and further limits the downstream applications of the device.
Accordingly, a goal of microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells. Microfluidic systems provide numerous advantages for single cell analysis: economies of scale, parallelization and automation, and increased sensitivity and precision that comes from small volume reactions. Considerable effort over the last decade has been directed towards developing integrated and scalable single cell genetic analysis on chip (7, 8). Thus, many of the basic functionalities for microfluidic single cell gene expression analysis have been demonstrated in isolation, including cell manipulation and trapping (9, 10), RNA purification and cDNA synthesis (11-13), and microfluidic qPCR (14) following off-chip cell isolation cDNA synthesis and preamplification. In particular, microfluidic qPCR devices (Biomark Dynamic Array, Fluidigm) have recently been applied to single cell studies (15, 16). Although these systems provide a high-throughput qPCR readout, they do not address the front end sample preparation and require single cell isolation by FACS or micropipette followed by off-chip processing and pre-amplification of starting template prior to analysis. The critical step of integrating all steps of single cell analysis into a robust system capable of performing measurements on large numbers of cells has yet to be reported. A single demonstration of an integrated device for directly measuring gene expression in single cells was described by Toriello et al., combining all steps of RNA capture, PCR amplification, and end-point detection of amplicons using integrated capillary electrophoresis (17). Despite the engineering complexity of this system, throughput was limited to four cells per run, cell capture required metabolic labeling of the cells, and the analysis was not quantitative.
Isolation of single or limited numbers of cells is required prior to many types of analysis and this typically requires the use of a cell trapping mechanism. Low trapping throughput and low trapping efficiency present a significant challenge to the goal of reliable and scalable analysis of single or small numbers of cells. Low capture efficiencies necessitating tens of thousands of cells in order to make a few single cell measurements is not an issue when using cell lines, however it becomes a significant problem when using primary samples of rare cell types, such as stem cells. Also, observations of both the trapped cells and those passing around the traps have indicated that the cell trapping efficiency was dependent on cell size, which could potentially introduce a bias into the single cell measurements.